It is usual in microscopy to generate image data of an object so that the object can be depicted on a display. Such image data can, for example, contain for each scanned object point an information item regarding the power level of detected light proceeding from that object point and/or regarding the wavelength of the detected light and information regarding the location of the respective object point, for example in the form of coordinate indications.
Three-dimensional scanning of an object is possible, for example, using a confocal microscope.
In scanning microscopy, a sample is illuminated with a light beam in order to observe detected light emitted from the sample as reflected or fluorescent light. The focus of an illumination light beam is moved in a sample plane with the aid of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in an X direction and the other in a Y direction. Tilting of the mirrors is brought about, for example, with the aid of galvanometer positioning elements. The power level of the detected light coming from the object is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the current mirror position.
In confocal scanning microscopy specifically, an object is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illumination light is coupled in via a beam splitter. The fluorescent or reflected light coming from the object travels back via the beam deflection device to the beam splitter, traverses it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that what is obtained is only a point information item that results, by sequential scanning of the object with the focus of the illumination light beam, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes are usually made up of a scanning module that is flange-mounted onto the stand of a conventional light microscope, the scanning module containing all the aforesaid elements additionally necessary for scanning a sample.
In confocal scanning microscopy, in the case of two-photon excitation (or multi-photon excitation) it is possible to omit a detection pinhole, since the excitation probability depends on the square of the photon density and thus on the square of the illumination intensity, which of course is much higher at the focus than in neighboring regions. The fluorescent light to be detected therefore derives with high probability in very large part from the focus region, which renders superfluous a further differentiation, using a pinhole arrangement, between fluorescence photons from the focus region and fluorescence photons from the neighboring regions.
Three-dimensional imaging, or the generation of image data that can permit three-dimensional depiction of the object, can also be accomplished with other types of microscopes. To be mentioned in this regard merely by way of example is SPIM technology, in which an object is transilluminated with a disk of light from different directions.
Ordinarily, a researcher would like to depict the object in such a way that its aspects that are important to him or her can easily be recognized. The user of a microscope, or the researcher who is evaluating image data obtained using a microscope, therefore needs to influence the manner of depiction, for example the size of the depiction of the object or the direction of view. In addition, especially in the context of a three-dimensional depiction, a need often exists to depict only specific parts of the objects and not other parts. It is often also desirable to be able to look in controlled fashion at a specific section plane of an object.